Infection is a major problem for the 5 to 6 million Americans with chronic wounds because it interferes with the normal healing process and increases the risk of serious complications that often necessitate limb amputation or result in death. A significant obstacle to the successful treatment of chronic wounds is the inability to correctly diagnose infection. Most clinicians still rely on visual signs and symptoms that are indicative of infection in acute or surgical wounds but have been proven to be inaccurate for diagnosis of infection in chronic wounds. The primary objective of this proposal is to optimize and validate a novel diagnostic test that will better enable physicians or nurses to detect chronic wound infection at the point-of- care. Our overall objective is produce a simple, single-use diagnostic device for hospitals, wound care clinics, outpatient centers, and long-term care facilities. The device will give a visible, yes-or-no result within 15 minutes when the bacterial concentration within the wound exceeds a level demonstrated to be indicative of infection. This would benefit public health by assisting individuals with chronic wounds to receive the appropriate treatment sooner, which will accelerate the healing process, relieve their suffering and discomfort, and lower the risk of infectious complications. Our Phase I objectives focused on the development of a functional diagnostic assay that was demonstrated to detect bacteria in chronic wounds with excellent diagnostic sensitivity and specificity relative to standard microbiological techniques. Our Phase II specific aims are focused on improvement and validation of the diagnostic test. The first aim is to evaluate a more accurate and reliable method of collecting tissue fluid from chronic wounds. This will help to reduce variability in the sample volume collected, which will improve diagnostic accuracy of the test. Our second objective is to change the assay readout from a quantitative ELISA format into a binary lateral flow-based test. An important step in this process is setting the appropriate threshold for the lateral flow line development using validated standards. Lastly, we plan to evaluate the diagnostic performance of test with samples from patients within the three major chronic wounds types: diabetic, pressure, and venous ulcers. Results of the diagnostic test will be compared to a reference standard for diagnosis of chronic wound infection to calculate the sensitivity and specificity. The data from this clinical study will evaluate the ability of the diagnostic test to detect chronic wound infection and will demonstrate clinical utility of the device. PUBLIC HEALTH RELEVANCE: The project goal is to develop a simple, inexpensive point-of-care diagnostic test to help clinicians rapidly determine if a chronic wound is infected. Earlier and more accurate infection diagnosis will allow patients to receive the appropriate treatment sooner, resulting in less pain and suffering and a lower risk of serious infection-related complications.